Cytoplasmic granules of cytotoxic T lymphocytes have been studied in order to assess their role in the lytic function of these cells. Granules from cloned CTL have been purified by Percoll gradient centrifugation and shown to contain two serine esterases capable of cleaving the trypsin substrate BLT in addition to the lytic cytolysin protein and a nuclear DNA releasing activity. The major BLT esterase in CTL is a 60kd protein, comprised of two 30kd protein chains linked by disulfide bonds. The enzyme is inactivated by diispropylfluorophosphage (DFP) and phenylmethylsulfonyl fluoride (PMSF), but these reagents react with the enzyme in intact cells inefficiently. This was shown to be caused by a low internal pH of the intact granules inside the cells, since agents known to raise the pH of acidic organelles (ammonia, chloroquine, monensin and nigericin) acted synergistically with PMSF in the inactivation of BLT esterase in intact cells but not the solubilized enzyme. The same selective synergistic effect was observed in the labeling of this protein band with radiolabelled DFP. When lytic function of CTL whose BLT esterase was greater than 95% inactivated by these treatments was examined, it was found to be virtually unaffected, indicating that the BLT esterase is probably not required for the CTL lytic process. The secretion of CTL BLT esterase into the medium has been shown to be a convenient marker for the granule exocytosis process. Using this assay, we have studied in vivo generated CTL from peritoneal exudates in which allo tumors have been recently rejected. Two directional allo-specific BLT esterase secretion has been demonstrated from such CTL, confirming the granule exocytosis mechanism operates in these cells during the lytic process. In addition to these studies with classical CTL, we have found that cloned helper T lymphocytes contain cytoplasmic granules with high levels of BLT esterase. At least some of these helper clones are capable of potent lytic function. We have found that BLT esterase is secreted into the medium by specific antigenic and MHC-restricted stimuli.